Summary RNA is an important therapeutic target, yet most RNA sequence-structure-function analysis is conducted in vitro rather than in the context of the cell despite substantive differences between the two environments. The objective of this proposal is to explore RNA sequence/function relationships within the cell using local fitness landscapes. Our model systems for this work are bacterial RNA regulators, such as riboswitches, which are antimicrobial targets and important biophysical models for studying RNA sequence-function relationships. Based on previous studies in the literature and our own preliminary data, we hypothesize that in vitro experiments imperfectly capture the sequence requirements required for biological function for many structured RNAs. To address how in vitro measured parameters correspond with cellular expression and organismal fitness, we plan to use three bacterial RNA regulators as model systems: the glycine riboswitch in Bacillus subtilis, the pyrR regulator in Streptococcus pneumoniae, and an FMN riboswitch in S. pneumoniae. Our specific aims are: 1) Determine which sequence changes to an RNA regulator impact organismal fitness and how these may be altered under different conditions that include defined culture medium, the presence of antibiotics, and a mouse infection model. 2) Establish the relationship between cellular gene expression profile and organismal phenotype. 3) Assess how gene expression and organismal fitness correspond with in vitro structure and function assays. We will achieve these aims by creating local fitness landscapes for each RNA regulator wherein all single and most double mutants are created as a pool, and subsequently evaluated using high-throughput sequencing as a final readout. The pools of variants will be appraised using a variety of techniques including: organismal competitions in culture (B. subtilis and S. pneumoniae), in the presence of targeting antibiotics (S. pneumoniae), and in a mouse infection model (S. pneumoniae); as well as microfluidic encapsulation of microcolonies to enable FADS-based assessment of cellular gene expression and in vitro transcription termination parameters. These high-throughput observations will be followed by assessment of a subset of mutants to confirm the observed phenotypes and examine secondary structure both in vitro and within the cell (SHAPE-MaP). Our proposal leverages organismal phenotypes we discovered in previous work and is an innovative approach to assess RNA sequence-function relationships that cannot be assessed in any other way. The expected outcome of the proposed work is the first mapping between RNA regulator sequence and a range of variables including organismal phenotype, gene expression, and in vitro termination characteristics. This contribution is significant because it will enable better interpretation of results from traditional in vitro and gene expression assays to inform efforts to target RNA regulators with antibiotics and to design novel RNA regulators.